Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial‐scale systems aiming at plasmid DNA purification.     

Activation of topoisomerase II during partial purification by heparin-Sepharose chromatography

Partial purification of topoisomerase II from small samples (10(7)-10(8) cells) of human leukaemic cells was achieved by isolation of cell nuclei, hyper-osmotic extraction of nuclear proteins, sorption of nuclear proteins by heparin-Sepharose and elution with potassium phosphate. Similar results were obtained by gradient and batchwise elution. The calatylic activity of topoisomerase increased ca. eightfold after removal of ca. 95% of the contaminating nuclear proteins. The conserved enzymatic activity after partial purification indicates that the enzyme was not damaged. The half-life of enzymatic activity is increased by the chromatographic procedure. Owing to its high yield and technical simplicity, this could be a candidate procedure for the study of topoisomerase II in patient-derived blood samples.     

Renewable microcolumns for automated DNA purification and flow-through amplification: from sediment samples through polymerase chain reaction

There is an increasing need for field-portable systems for the detection and characterization of microorganisms in the environment. Nucleic acids analysis is frequently the method of choice for discriminating between bacteria in complex systems, but standard protocols are difficult to automate and current microfluidic devices are not configured specifically for environmental sample analysis. In this report, we describe the development of an integrated DNA purification and polymerase chain reaction (PCR) amplification system and demonstrate its use for the automated purification and amplification of Geobacter chapellei DNA (genomic DNA or plasmid targets) from sediments. The system includes renewable separation columns for the automated capture and release of microparticle purification matrices, and can be easily reprogrammed for new separation chemistries and sample types. The DNA extraction efficiency for the automated system ranged from 3 to 25%, depending on the length and concentration of the DNA target. The system was more efficient than batch capture methods for the recovery of dilute genomic DNA even though the reagent volumes were smaller than required for the batch procedure. The automated DNA concentration and purification module was coupled to a flow-through, Peltier-controlled DNA amplification chamber, and used to successfully purify and amplify genomic and plasmid DNA from sediment extracts. Cleaning protocols were also developed to allow reuse of the integrated sample preparation system, including the flow-through PCR tube.     

Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels

The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 microm) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)6-tagged single chain (sc) Fv antibody fragments, (His)6-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)6-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques.     

Current trends in separation of plasmid DNA vaccines: A review

Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.     

Isolation and identification of the glucuronide conjugate of 2-hydroxydesipramine by preparative liquid chromatography

A semi-preparative column liquid chromatographic procedure for the isolation and purification of milligram quantities of the glucuronide conjugate of 2-hydroxydesipramine, a major metabolite of desipramine, is presented. Urine from patients receiving desipramine was collected and passed through a column of XAD-2 resin. The methanolic extract was chromatographed on a reversed-phase octadecyl semi-preparative column followed by further purification on a silica gel column of the same dimension, yielding a product 95% pure. Fast atom bombardment and thermospray mass spectroscopy, as well as ultraviolet photodiode-array spectroscopy and hydrolysis with beta-glucuronidase confirmed the identification and purity of 2-hydroxydesipramine glucuronide. This important glucuronide metabolite will be a useful tool as an authentic standard for pharmacokinetic and metabolism studies and for determining its pharmacological characteristics in laboratory animals.     

Isolation of tomato pectin methylesterase and polygalacturonase on monolithic columns

An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 microg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 microg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.     

Purification of bacilli ribonucleases by reversed-phase high-performance liquid chromatography

A two-step purification method is presented that utilizes the specific chromatographic properties of bacilli extracellular cycling ribonucleases. A double gradient system of elution is used. Initial concentration of the sample followed by reversed-phase HPLC gives high yields (90–95%) of homogeneous, active protein on both analytical and preparative scales. The procedure may be applied to the isolation of ribonucleases from different sources without significant modifications     

Apolar chromatography on Sephadex LH-20 combined with high-speed counter-current chromatography. High yield strategy for structurally closely related analytes-Destruxin derivatives from Metarhizium anisopliae as a case study

A novel high yield isolation procedure for lipophilic cyclic peptide derivatives is presented. Destruxin (dtx) A, B, D, E, and E-diol retrieval from Metarhizium anisopliae culture broth was achieved with a three-step purification protocol. After liquid-liquid extraction column chromatography over Sephadex LH-20 served as enrichment step. High-speed counter-current chromatography (HSCCC) was used for the final purification. Within the first chromatographic step dtx D and dtx E-diol were separated in purities exceeding 90%. The separation of dtx A, B, and E was achieved from an enriched Sephadex LH-20 fraction by a HSCCC protocol using light petroleum-ethyl acetate-methanol-water = 2:5:2:5 (v/v) as eluent system. These derivatives were obtained in purities above 98% and total yields exceeding 40%.     

碳纳米管的色谱纯化法

目前碳纳米管的纯化方法很多,有物理法,化学法和物理化学综合法等,然而这些方法仍不能得到高纯度的碳纳米管。色谱法分离纯化碳纳米管已成为近年来研究的热点,该法不仅能高效分离得到高纯度的碳纳米管,而且还能达到长短分离,具有重要的学术和实际意义。已报道的色谱法纯化碳纳米管主要有空间排阻色谱法以及毛细管电泳法,本文对上述色谱法纯化碳纳米管做了详细介绍。     

Isolation and characterization of proteoglycans from different tissues

Two chromatographic procedures for the isolation and purification of proteoglycans (PG) and their related glycosaminoglycan (GAG) peptides are described. PG from human aorta were isolated from tissue extract by sequential ion-exchange, size-exclusion and hydroxyapatite chromatography. Final purification of samples was achieved by chromatography on Mono Q. Homogeneity of samples was demonstrated by Western blot analysis of biotin-labelled compounds prior to and after enzymatic digestion and dual-wavelength detection in size-exclusion chromatography. The purity of samples obtained by the procedure described was sufficient for protein sequence analysis. GAG preparations of bovine trachea cartilage were purified by the sequential use of strong anion-exchange supports. Molecular weight distribution and sensitivity to treatment with glycan-specific enzymes was shown by size-exclusion chromatography.     

Multi-stage chromatographic procedure for the isolation of a single oligomeric species (vinyl chloride tetramer) from poly(vinyl chloride) resin used for food packaging applications

A multi-stage chromatographic procedure is described whereby low-molecular-weight material initially obtained by soxhlet diethyl ether extraction from food grade poly(vinyl chloride) (PVC) resin, is fractionated to yield firstly a mixture of vinyl chloride oligomers and then a single species free from other impurities. A number of column chromatographic procedures were evaluated, and a combination of high-performance size exclusion columns using initially tetrahydrofuran solvent followed by toluene solvent were shown to produce the most effective separation. The isolation and purification of a vinyl chloride tetramer was monitored by capillary column gas chromatography with specific chlorine detection (Hall electrolytic conductivity detector) and the identification confirmed by mass spectrometry. Using this newly developed procedure 0.5 mg of vinyl chloride tetramer was isolated from a PVC base resin.     

An Efficient and Easy Method for the One‐pot Synthesis of Spirooxindoles in the Presence of Na2CO3

An efficient and easy procedure for the one‐pot synthesis of spirooxindoles in the presence of Na₂CO₃ in water : methanol (1:1) is described. The salient features of this method are simple methodology, shorter reaction time, easy product isolation, and no chromatographic purification.     

Purification of alkaloids from Corydalis yanhusuo W. T. Wang using preparative 2‐D HPLC

Two‐dimensional preparative multi‐channel parallel high performance liquid chromatography was successfully applied for the first time to isolate and purify alkaloids from Corydalis yanhusuo. The experiments were performed in off‐line mode using the same preparative chromatographic column with pH 3.5 in the first and pH 10.0 in the second separation dimension. In the preparative process, UV‐triggered fraction collection was used in the first dimension while UV and MS‐triggered collection were used in the second dimension for reasons of sensitivity and complementarity. Two pure compounds and nine fractions were obtained in the first dimension. Then two representative fractions were further purified in the second dimension and six pure compounds were obtained. The results demonstrated that this procedure is an effective approach for the preparative isolation and purification of alkaloids from Corydalis yanhusuo. Based on the different pH values of the mobile phase in this method, it is also suitable for the preparative isolation and purification of other compounds from TCMs which are sensitive to the pH of the solutions. Moreover, this method will be a promising tool for the purification of low content compounds from natural products.     

油菜籽中主要硫甙的分离提纯

用柱色谱方法从甘蓝型油菜籽中分离提纯了1-硫［(1Z)-3-羟基-1-［(磺酸基)亚氨基］-4-戊烯基］-1-硫代-β-D-葡萄糖钾盐(progoitrin)。用甲醇溶液提取菜籽中的硫甙,得到粗提物;粗提物经酸性氧化铝色谱柱与反相C18硅胶柱进一步分离提纯,得到纯品。对纯品进行紫外光谱、红外光谱、核磁共振氢谱、质谱和元素分析,测得的数据与文献值相符。用高效液相色谱测得硫甙提取物的纯度为99%。该方法操作简便,得到的硫甙样品纯度高,是一种有价值的硫甙提取方法,具有良好的应用前景。     

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.     

Purification of human 6-phosphogluconate dehydrogenase from human haemolysate with chromatography on an immobilized dye as the essential step and use of automation. Simultaneous purification of lactate dehydrogenase

The screening procedure described in the preceding paper allowed a practical purification procedure to be devised that was automated for human 6-phosphogluconate dehydrogenase. The purification needed only two chromatographic steps, first on immobilized Procion Blue HE-GN and then on Phenyl-Sepharose. This technique also gave purified lactate dehydrogenase. Both enzymes showed single bands in SDS polyacrylamide gel electrophoresis.     

A modified protocol for isolation and purity evaluation of a staphylococcal acidic polysaccharide by chromatography and capillary electrophoresis

The extracellular slime of Staphylococcus epidermidis contains, amongst various macromolecules, an acidic polysaccharide (PS) of a molecular mass of 20 kDa with significant antigenic and biological properties. The isolation procedure used so far includes multiple fractionations in anion‐exchange chromatographic columns before its final purification by gel filtration chromatography. This protocol is laborious, time‐consuming and includes the risk of unnecessary loss of PS quantities. Because of the significance of this PS, a modified protocol resulting in an easier and quicker isolation procedure was developed. Furthermore, identification, purity, charge density and molecular integity of the isolated polysaccharide were evaluated by a reverse‐polarity capillary electrophoresis method. Copyright © 2010 John Wiley & Sons, Ltd.     

Sample preparation and isolation using planar chromatographic methods

The state of art of the various preparative planar liquid chromatographic (PLC) methods is summarized, especially for off-line and on-line sample application. The sample purification possibilities for PLC techniques are discussed. Purification and isolation strategies using forced-flow planar chromatographic techniques, such as overpressured layer chromatography and rotation planar chromatography, are suggested in the form of flow charts.     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。